Integrated Functions of Pax3 and Pax7 in the Regulation of Proliferation, Cell Size and Myogenic Differentiation

Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed

240th ENMC workshop: the involvement of skeletal muscle stem cells in the pathology of muscular dystrophies 25 -27 January 2019, Hoofddorp, the Netherlands

Satellite cells are dysfunctional in several neuromuscular disorders. Some muscles are more susceptible than others to disease and ageing. In vitro and in vivo model systems have shed light on many of the processes involved. in satellite cell function and dysfunction, but the drawbacks of each model system must be considered. Skeletal muscle pathology in mouse models of neuromuscular disease are affected by genetic background. A single cell approach will be useful to identify dysfunction in subsets of cell populations

Advances in skeletal myogenesis: from molecular regulation to cell biology and pathology

International audienc

From cyclins to CDKIs: Cell cycle regulation of skeletal muscle stem cell quiescence and activation

International audienc

The Notch signaling network in muscle stem cells during development, homeostasis, and disease

International audienceAbstract Skeletal muscle stem cells have a central role in muscle growth and regeneration. They reside as quiescent cells in resting muscle and in response to damage they transiently amplify and fuse to produce new myofibers or self-renew to replenish the stem cell pool. A signaling pathway that is critical in the regulation of all these processes is Notch. Despite the major differences in the anatomical and cellular niches between the embryonic myotome, the adult sarcolemma/basement-membrane interphase, and the regenerating muscle, Notch signaling has evolved to support the context-specific requirements of the muscle cells. In this review, we discuss the diverse ways by which Notch signaling factors and other modifying partners are operating during the lifetime of muscle stem cells to establish an adaptive dynamic network

Pax7 haploinsufficiency impairs muscle stem cell function in Cre-recombinase mice and underscores the importance of appropriate controls

International audienceAbstract Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies

Extraction and sequencing of single nuclei from murine skeletal muscles

International audienceSingle-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications. For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021)

Reciprocal signalling by Notch–Collagen V–CALCR retains muscle stem cells in their niche

International audienceThe cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1,2,3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4,5,6,7,8,9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch–COLV–Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations

Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers

International audienceSkeletal muscle fibers are large syncytia but it is currently unknown whether gene expression is coordinately regulated in their numerous nuclei. Here we show by snRNA-seq and snATAC-seq that slow, fast, myotendinous and neuromuscular junction myonuclei each have different transcriptional programs, associated with distinct chromatin states and combinations of transcription factors. In adult mice, identified myofiber types predominantly express either a slow or one of the three fast isoforms of Myosin heavy chain (MYH) proteins, while a small number of hybrid fibers can express more than one MYH. By snRNA-seq and FISH, we show that the majority of myonuclei within a myofiber are synchronized, coordinately expressing only one fast Myh isoform with a preferential panel of muscle-specific genes. Importantly, this coordination of expression occurs early during post-natal development and depends on innervation. These findings highlight a previously undefined mechanism of coordination of gene expression in a syncytium